The present invention relates to a method of preserving sperm to be used for the artificial insemination of domestic animals, and especially for the artificial insemination of swine and bovine. The invention can also find application in avian, other domestic animals and human sperm.
The most common method of sperm preservation is cryopreservation. During the process of cryopreservation, freezing and thawing damage cell membranes. Mammalian sperm are sensitive to rapid cooling. This phenomenon, called cold shock, occurs when the environmental temperature of sperm rapidly decreases from 35-37xc2x0 C. (body temperature) down to a few degrees above zero (Watson and Plummer, 1985). For boar sperm, even slow cooling below a temperature of +15xc2x0 C. results in a decreased survival rate (Watson and Plummer, 1985). Cryopreserved boar semen is used on a very small scale for artificial insemination because of reduced fertility compared to fresh sperm (Johnson and Larson, 1985; Hofmo and Almlid, 1991; Bwanga, 1991). Many experiments (reviewed by Watson and Plummer, 1985) attribute this poor performance to the detrimental effect of cryopreservation on the sperm membrane.
Sperm injury is manifested as loss of selective permeability and loss of integrity of the plasma membrane, outer acrosomal membrane, and mitochondria (reviewed by Parks and Lynch, 1992). These manifestations are accompanied by loss of motility, decreased energy production, changes to membrane lipid composition (Parks and Lynch, 1992; Buhr et al., 1994) and changes to membrane dynamic behaviour (Buhr et al., 1989; 1994). In order to use sperm for artificial insemination, there is a need to prevent and repair loss of selective permeability and loss of integrity of the plasma membrane, outer acrosomal membrane, and mitochondria. There is a need to characterize the damage to sperm during cryopreservation. There is also a need to develop compositions and methods that may be used to prevent and repair the damage to sperm during cryopreservation so that survival of sperm may be increased.
Susceptibility of sperm to cryopreservation differs across species and with stage of spermatozoal maturation. It could be partially due to the different lipid composition of sperm membranes as evidenced by: 1. Sperm from different animal species with similar cold shock resistance, have rather similar lipid composition, length and saturation of fatty acid chains (Watson and Plummer, 1985; Parks and Lynch, 1992); 2. Sperm from different parts of the epididymis have different sensitivity to cold shock, which is correlated with the changes in lipid composition during sperm maturation from caput to cauda epididymis (Bwanga, 1991); 3. Some lipids are released from sperm membranes during cold shock (Darin-Benneett et al., 1973; Bwanga, 1991); 4. The main protective factor from egg-yolk is crude lipid which has been demonstrated to interact with sperm plasma membrane (Gebauer et al., 1970; Pursel et al., 1972; Watson, 1975; Foulkes, 1977); and 5. Addition of some lipids to the extender has been suggested to have beneficial effects on cold resistance (Paquignon, 1985). These compositions have not significantly improved boar sperm viability after cryopreservation.
The nature and amount of specific lipids in sperm membranes differ among animal species (Darrin-Bennett et al., 1974; Poulos et al., 1973; Parks and Lynch, 1992: Buhr et al., 1994), which is consistent with the species"" differences in the susceptibility of sperm to cold shock (Watson and Morris, 1981; Watson and Plummer, 1985). Sperm from rooster (Parks and Lynch, 1992; Watson, 1981), human and monkey (Holt and North, 1985), rabbit and dog (reviewed by Watson and Plummer, 1985) are more resistant to cold shock than that from domestic animals, such as bull, ram, horse and boar, with boar sperm being the most sensititive (Watson, 1981). The results from Parks and Lynch (1992) showed that the ratio of sperm membrane proteins to phospholipids (wt:wt) was lowest for rooster at 0.46, intermediate for bull and stallion (0.80 and 0.86) and highest for boar (1.26). They also found that the ratio of cholesterol to phospholipid was close to 1 in human and monkey, but less than 0.8 in bull, ram and boar (Holt and North, 1985).
Using exogenous lipid as a cryoprotectant in semen cryopreservation has been tried by several groups (Butler and Roberts, 1975; Streiner and Graham, 1987; Wilhelm et al., 1996). However, a consistent improvement of post-thaw result has not been achieved, especially with boar semen. These problems could possibly be due a failure to meet a necessity for: 1. Lipids specific for different animal species. Experimental addition of phosphatidylcholine (PC), phosphatidylserine (PS) and/or cholesterol (Pursel et al., 1973; Paquignon, 1985) was based merely on the observation that egg-yolk lipoprotein has some beneficial effects on survival of cryopreservation. 2. Specific fatty acid chain length and saturation, and/or 3. Appropriate methodology to incorporate lipids into spermatozoa and monitor the incorporation efficiency.
Lipids considered common to the cell membrane such as phosphoglycerides, sphingomyelin (SPH) and cholesterol have been identified in the spermatozoa of a variety of species (Darin-Bennet et al., 1973, 1974; Darin-Bennet and White, 1977). Generally, PC is the predominant phospholipid in sperm membranes. Sphingomyelin and phosphtidylethanolamine (PE) are relatively high also. Phosphatidylserine and phosphatidylinositol (PI) are present at low levels. Lipid composition of whole sperm or isolated membranes has been shown to change during epididymal maturation in a variety of species (Nikolopoulou et al., 1985; Hall et al., 1991; Rana et al., 1991). Sperm lipids also change during capacitation and the interaction with ova (Nikolopoulou et al., 1986; Stojanoff et al., 1988; Seki et al., 1992).
Comparing sperm from domestic species, boar sperm membranes have a low percentage of PC and higher percentage of PE and SPH (De Leeuw et al., 1990), and the PI of boar is about 3 times of bull. Parks and Lynch (1992) found that bull and rooster sperm were characterized by a high ratio of PC to PE while boar and stallion sperm had a lower PC/PE ratio. Rooster sperm were also characterized by a higher percentage of phospholipid in the PS+PI fraction than other species (Parks and Lynch, 1992).
The composition of the acyl side chains of phospholipids also differs among species. Sperm membranes from cold-shock resistant species are characterized by a high degree of saturation in fatty acid chains (Darin-Bennet and White, 1977). Cold-shock susceptible species tend to have only very minor amounts of other aldehydes present, while resistant species contain large amounts of steraldehyde (18:0) and 16:1, 18:1, 18:2 aldehydes (Poulos et al., 1973; Darin-Bennett et al., 1974). Phosphatidylcholine from mammalian sperm is characterized by a very high proportion of docosapentanoyl and docosahexanoyl chains, with 22:5 predominant in boar and stallion sperm and 22:6 highest in bull sperm (Parks and Lynch, 1992). For PE, high proportion of long chain polyunsaturated fatty acyl groups, especially 22:5 and 22:6, were contained in sperm of bull, boar, stallion and ram while 22:4 was high in rooster sperm (Parks and Lynch, 1992). These results suggest that the lipid composition of sperm membranes contributes to the cold-shock sensitivity. There is a need to use information regarding the lipids and fatty acids which are damaged during cryopreservation in designing compositions for preserving semen. It would be helpful if a composition could be developed which protected and restored the specific kinds and ratio of lipids and fatty acids sperm membrane in order to improve cold resistance. There is currently no composition for improving sperm survival after cooling which is tailored to the kinds and ratio of lipids and fatty acids in sperm membrane.
Cryopreservation decreases the fertility of sperm, especially boar sperm, by reducing motility and damaging membrane integrity (Hofmo and Almlid, 1991). Changes in boar sperm membrane structure and function are manifested as dramatic alterations to both the composition and the dynamic behaviour of the lipids from sperm head plasma membranes (HPM) (Buhr et al., 1994). Modified ultrastructure was also found in boar sperm plasma membranes after cold shock (De Leeuw et al., 1990), which may affect the processes of capacitation, acrosome reaction and gamete recognition/fusion. Buhr et al. (1994) demonstrated for the first time that composition of lipids from the head plasma membrane of intact boar sperm was altered after cryopreservation. These compositional changes were correlated with significant fluidity changes in the response of the extracted lipids to temperature and calcium.
Compared to fresh sperm, cryopreserved boar sperm contained significantly less SPH and more PC (Buhr et al., 1994). The octadecanoate (18:0) content in both PC and PE decreased after cryopreservation, while the polyunsaturated fatty acids docosatetraenoate (22:4) and/or arachidonate (20:4) increased in these phospholipids and in SPH and PI. The alterations in the molecular interactions, composition, and Ca++ sensitivity of membrane lipids may disturb the normal behaviour of membranes in the fertilization process (Buhr et al., 1994).
Cryopreservation irreversibly affects membrane mechanics, especially the lateral phase separation of membrane lipids into fluid and gel phase domains. Isolated HPM from fresh boar sperm extended in BL-1, showed a decrease in fluidity over time (Buhr et al., 1989, Robertson et al., 1990). Thus, while the decrease in fluidity over time is similar for HPMs from fresh and untreated and fresh BL1-extended semen, sperm which have been extended and cooled, frozen-thawed, or cold-shocked show different membrane fluidity patterns. As the temperature decreases, fatty acyl chains of phospholipids become rigid and phospholipids become isolated in gel domains. Membrane proteins are excluded from gel domains (Pringle and Chapman, 1981) which results in destabilization of the membrane (Flechon et al., 1986). Thus changes to membrane constituents which affect the fluidity and stability of the membrane will affect the functioning of that membrane. This harmful effect is irreversible in sperm and the techniques and extenders currently employed have not prevented this damage. Exposure then, to cold temperatures prior to cryopreservation affects the membrane""s ability to regulate itself and other cellular functions, even in the presence of a cryoprotectant.
Cold-shocking sperm is also associated with the release of phospholipids from the cell membranes (Darin-Bennett et al., 1973). Although the release of total phospholipid in boar sperm was less than that seen in either bull or ram, PC and PE were preferentially released (Darin-Bennett et al., 1973). It was implied that these phospholipids were released from the acrosomal membrane, but HPM might also be involved (Bwanga, 1991). Using exogenous lipid as a cryoprotectant in semen cryopreservation has been tried by several groups (Butler and Roberts, 1975; Streiner and Graham, 1987; Wilhelm et al., 1996). However, a consistent improvement of post-thaw result has not been achieved, especially with boar semen. There is a need for a composition which increases boar sperm survival after chilling. There is a particular need for a composition which is designed to prevent and repair the damage specific to boar sperm membranes by providing lipids with specific fatty acid chain length and saturation of the lipids that are damaged by chilling of boar sperm. It would also be helpful if this composition could be used with methodology to incorporate lipids into spermatozoa and monitor the incorporation efficiency.
The invention comtemplates that liposome-cell interaction is important in sperm preservation. Phospholipids, in the presence or absence of the other amphipathic molecules such as cholesterol, typically form closed membranous vesicles when exposed to aqueous media. Small unilamellar vesicles (SUVs) are typically prepared by sonication to break up the multilamellar vesicles (MLV) (Huang, 1969). Typically, SUVs are a homogeneous population of 25 to 50 nm (Deamer and Uster, 1985, Blumenthal et al., 1977)). Theoretically, SUVs are prone to fusion, particularly at the phase transition temperature (Huang, 1983; Jones and Chapman, 1995). SUVs are more fusogenic, because they have higher surface tension due to the greater radius of curvature and because they can approach closer to the cell surface due to their small size (Huang, 1983).
Fusion efficiency can be defined generally as the occurrence of fusion or, specifically, as the extent of liposome fusion to target cells. Fusion efficiency can be determined semi-quantitatively by analysing the change of fluorescent intensity of single labelled fluorescence marker (Kok and Hoekstra, 1992) or by energy transfer between double labelled fluorescence markers (Struck et al., 1981). There are several methods for studying liposome-cell interactions (Huang, 1983), such as uptake of radioactive markers, carboxyl fluoroscein, fluorescent lipids, electron microscopy, R18 dequenching, resonance energy transfer. The method of the invention used one semi-quantitative method, either resonance energy transfer (RET) and R18 Dequenching, plus the quantitative flow cytometric technique to monitor fusion between SUVs and sperm membrane.
This invention provides compositions that incorporate specific exogenous lipids into sperm to improve their ability to survive cryopreservation. The compositions consist of lipids to be incorporated into the sperm. While other researchers have attempted to alter the molecular composition of sperm membranes, their additions have been based on assumptions about what a membrane may need in order to function better. We have taken a unique approach of: defining the particular problemxe2x80x94cryopreservation damages the fertilising ability of sperm by at least one mechanism not connected with sperm motility; specifying and then quantifying the specific molecules damaged in cryopreserved sperm; designing a unique mixture of phospholipids with specific fatty acid side chains; developing a method to fuse these lipids to sperm and quantitate the percentage of sperm taking up the lipids; demonstrating the enhanced resistance to chilling injury in lipid-treated sperm. These lipids were designed based on the damage we identified in cryopreserved boar sperm, which have unique membrane molecular composition, chilling sensitivity and functional parameters (calcium flux, capacitating conditions, zona penetration rate etc) when compared to all other domestic species. The same method is applied to bull sperm, which because of these differences, is not an obvious combination. The demonstrated efficacy of the lipids in bull sperm suggests that they may have universal efficacy in improving the post-thaw quality of cryopreserved sperm from many species.
The compositions also form stable SUVs under the normal physiological conditions for sperm and fuse with the sperm membranes. We also defined the incorporation conditions and fusion efficiency of selected lipids to boar and bovine sperm. An appropriate monitoring system was selected. Finally, we examined the effects of these lipids on viability and motility of boar and bovine sperm during the cooling procedure and after cryopreservation.
There is industrial utility. For example, in the bull industry, the enhanced sperm survival will mean that fewer sperm will be needed to achieve the current fertility success rates, and the industry therefore will be able to prepare more inseminating doses from the same number of sperm. In addition, bulls of high genetic merit and in strong commercial demand sometimes produce sperm which cannot survive the freezing and thawing process, and so the industry is unable to market this semen. The lipids will improve these sperm to allow meeting this established market demand. In the porcine artificial insemination industry, no current cryopreservation procedure is sufficiently efficient to allow widespread commercial use of frozen sperm. An effective freezing method will facilitate the development of a new international commercial trade in porcine genetics. Many species (domestic, exotic and endangered, including mammals and avians) have sperm that cannot survive cryopreservation, and these lipids will enable the successful preservation of this genetic material. The application of these lipids to enable successful preservation of poor quality sperm from individual males may find human medical application, and/or application in many other species.
The invention relates to a composition for increasing sperm survival, comprising a carrier, phospholipids and fatty acid chains. The invention also relates to a composition for increasing sperm survival, comprising a carrier, phospholipids and fatty acid chains, the composition being essentially absent of cholesterol. The composition can consist of phospholipids selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, phosphatidylserine and phosphatidylinositol. The composition preferably has phospholipids in a ratio where phosphatidylcholine is about 20: phosphatidylethanolamine is about 25: sphingomyelin is about 40: phosphatidylserine is about 5: phosphatidylinositol is about 5. More preferably, the composition has phospholipids in a ratio where phosphatidylcholine is about 21: phosphatidylethanolamine is about 26: sphingomyelin is about 42: phosphatidylserine is about 5: phosphatidylinositol is about 5. Most preferably, the composition has the phospholipids in a ratio where phosphatidylcholine is about 21.05: phosphatidylethanolamine is about 26.32: sphingomyelin is about 42.11: phosphatidylserine is about 5.26: phosphatidylinositol is about 5.26. In another preferred embodiment, the phospholipids are in a ratio where phosphatidylcholine is about 20: phosphatidylethanolamine is about 20: sphingomyelin is about 40: phosphatidylserine is about 10: phosphatidylinositol is about 10. Alternatively, the the phospholipids are in a ratio where phosphatidylcholine is about 25 : phosphatidylethanolamine is about 20 : sphingomyelin is about 40: phosphatidylserine is about 5 : phosphatidylinositol is about 10.
The composition preferably has the phospholipids having proportion (mol %) of:
a. phosphatidylcholine with specific fatty acid chains about:
C16: 0=7%
C18: 0=3%
C18: 1=5%
C18: 2=4%
C20: 4=0.8%
C22: 6=0.6%
b. phosphatidylethanolamine with specific fatty acid chains about:
C16: 0=5%
C18: 0=21%
c. sphingomyelin with specific fatty acid chains about:
C16: 0=16%
C18: 0=25%
C22: 6=5%
d. phosphatidylserine with specific fatty acid chains about:
C16: 0=0.5%
C18: 0=4%
C18: 2=0.5%
e. phosphatidylinositol with specific fatty acid chains about:
C16: 0=0.6%
C18: 0=6%
C18: 1=0.2%
C18:2=0.6%,
In another embodiment, the phospholipids in the composition have a proportion (mol %) of
a. phosphatidylcholine with specific fatty acid chains about:
C16: 0=7.37%
C18: 0=3.16%
C18: 1=5.26%
C18: 2=3.79%
C20: 4=0.84%
C22: 6=0.63%
b. phosphatidylethanolamine with specific fatty acid chains about:
C16: 0=5.26%
C18: 0=21.06%
c. sphingomyelin with specific fatty acid chains about:
C16: 0=15.97%
C18: 0=25.27%
C22: 6=5.34%
d. phosphatidylserine with specific fatty acid chains about:
C16: 0=0.53%
C18: 0=4.21%
C18: 2=0.53%
e. phosphatidylinositol with specific fatty acid chains about:
C16: 0=0.62%
C18: 0=6.22%
C18: 1=0.20%
C18:2=0.62%.
The phospholipids in the composition may have the phospholipids in one of the ratios described above and the fatty acid side chains in another of the ratios described above. The sperm preserved with the compositions is preferably mammalian sperm, most preferably boar sperm or bull sperm.
The compositions preferably have fatty acid chain proportions that imitate the fatty acid chain proportions found in fresh boar sperm membrane or fresh bull sperm membrane.
The compositions preferably have fatty acid chain proportions that imitate the fatty acid chain proportions found in fresh boar sperm membrane with adjustments to broaden the distribution of fatty acid chains in phosphatidyicholine. The fatty acid chain proportions may also imitate the fatty acid chain proportions found in fresh boar sperm membrane with adjustments to increase the proportion of 18:0. The fatty acid chain proportions also preferably imitate the fatty acid chain proportions found in fresh boar sperm membrane with adjustments to decrease or increase the proportion of longer unsaturated fatty acids or unsaturated fatty acids.
The fatty acid chains are preferably in about the following percentages: phosphatidylcholine 35% 16:0, 15% 18:0, 25% 18:1, 18% 18:2, 4% 20:4 and 3% 22:6. phosphatidylethanolamine 20% 16:0, 80% 18:0 and no 20:4, 22:4 and 22:6, sphingomyelin 30% 16:0, 60% 18:0, 10% 22:6 and no 20:4 or 22:4, phosphatidylserine 10% 16:0, 80% 18:0, 10% 18:2 and phosphatidylinositol 28% 16:0, 65% 18:0, 2% 18:1 and 5% 20:0.
The compositions of the invention increase sperm survival during cooling, freezing and post-thaw. The phospholipids and fatty acid chains are preferably in a vesicle, more preferably a small unilamellar vesicle (SUV). The compositions of the invention may be combined with an extender, preferably BTS, BF5, egg yolk or O.E.P. The composition of any of claims 1 to 22, wherein phospholipids and fatty acid chains are selected in proportions based on differences in membrane lipids caused by cryopreservation.
The invention also includes sperm preserved with the compositions of the invention. The invention includes a method for increasing sperm survival comprising characterizing the damage caused to sperm lipids upon cooling, freezing or thawing and administering an exogenous composition containing lipids of the type that are damaged upon cooling, freezing or thawing. In the method, the exogenous lipid composition is preferably administered in a vesicle, more preferably a small unilamelar vesicle (SUV).
The invention also includes a method for increasing sperm survival comprising administering one of the compositions of the invention.
In another embodiment, the invention includes a method of preparation of a composition for preserving sperm for in vitro or in vivo fertilization, comprising: determining and quantifying at least one of the phospholipids damaged in chilled, frozen or post-thaw sperm; preparing a composition including a carrier and at least one phospholipid with fatty acid side chains to replace at least one phospholipid damaged in chilled or frozen sperm, wherein the composition is capable of fusing to sperm to provide the sperm with resistance to chilling, freezing or post-thaw damage. The invention also includes a method of preparation of a composition for preserving sperm for in vitro or in vivo fertilization, comprising: determining and quantifying the phospholipids damaged in chilled, frozen or post-thaw sperm; preparing a composition including a carrier and phospholipids with specific fatty acid side chains to replace the phospholipids damaged in chilled or frozen sperm, wherein the composition is capable of fusing to sperm to provide the sperm with resistance to chilling, freezing or post-thaw damage. In the method, the sperm is mammalian sperm, more preferably boar sperm or bull sperm.
The invention also includes a method of performing artificial insemination in a mammal comprising administering sperm preserved with the composition of the invention to a female mammal of the same species so that the female mammal becomes impregnated with the sperm. The mammal is preferably a bovine or porcine mammal.